Pockwinse Shirwin M, Rajgopal Arun, Young Daniel W, Mujeeb Khwaja A, Nickerson Jeffrey, Javed Amjad, Redick Sambra, Lian Jane B, van Wijnen Andre J, Stein Janet L, Stein Gary S, Doxsey Stephen J
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
J Cell Physiol. 2006 Feb;206(2):354-62. doi: 10.1002/jcp.20469.
RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies.
RUNX/AML转录因子是多种细胞系中细胞生长和分化的关键调节因子，与包括急性髓性白血病（RUNX1）、乳腺癌（RUNX2）和胃癌（RUNX3）在内的人类癌症相关。RUNX蛋白通过特定的核内亚定位信号靶向细胞核内的基因调控微环境。然而，人们对RUNX在细胞质和细胞核之间的分布及区室化动态了解甚少。在此，我们通过免疫荧光显微镜观察发现，当化疗药物紫杉醇使微管稳定时，RUNX2从细胞核重新定位到细胞质。RUNX2依赖紫杉醇的细胞质积累受到雷帕霉素B的抑制，雷帕霉素B可阻断CRM-1依赖的核输出，且不受蛋白质合成抑制剂环己酰亚胺的影响。通过生化分析，我们发现内源性RUNX2以浓度依赖的方式与稳定的微管结合，且RUNX2的氨基末端介导微管结合。在细胞的可溶部分，RUNX2与α微管蛋白共免疫沉淀，表明微管结合涉及α/β微管蛋白亚基。我们得出结论，RUNX2与微管结合并在细胞核和细胞质之间穿梭。我们提出，RUNX2的核质穿梭可能会调节其转录活性，以及其与整合在含有RUNX2的亚核位点的信号转导途径相互作用的能力。紫杉醇诱导的RUNX2核水平和/或其他细胞生长调节因子的急性耗竭可能代表了紫杉醇在癌症化疗期间发挥其生物学效应的另一种途径。
